EP1417488B1 - Zellwanderungsassay - Google Patents

Zellwanderungsassay Download PDF

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EP1417488B1
EP1417488B1 EP02739361A EP02739361A EP1417488B1 EP 1417488 B1 EP1417488 B1 EP 1417488B1 EP 02739361 A EP02739361 A EP 02739361A EP 02739361 A EP02739361 A EP 02739361A EP 1417488 B1 EP1417488 B1 EP 1417488B1
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cell migration
ligand
chemokine
receptor
antagonist
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EP1417488A4 (de
EP1417488A2 (de
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Zheng Wei
Zhenhua Miao
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Chemocentryx Inc
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Chemocentryx Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention is directed to an assay for identifying antagonists of chemoattractant receptors, such as chemokine receptors.
  • chemoattractant receptors such as chemokine receptors.
  • One advantage of the assay compared with prior assays is its ability to discriminate valid chemoattractant receptor antagonists from those compounds that generate false positive and negative signals.
  • High-throughput screening (HTS) methods for identifying antagonists of chemoattractant receptors often rely on detecting perturbations in downstream events, such as cell migration.
  • chemokine receptors In the case of chemokine receptors, leukocyte cell migration is often assayed.
  • compounds disrupting cell membranes or blocking downstream events mimic these outcomes, masquerading as candidate antagonists.
  • Considerable effort is then required to distinguish the genuine antagonists from those compounds or molecules that caused false positive signals. Identifying true antagonists, which represent only a very small fraction of the large collections of candidate antagonists analyzed in high-throughput screens, is a daunting task. Realizing any savings in time or expense can bring a new drug to patients more quickly and less expensively.
  • Small molecules that initially appear to be inhibitors of receptor-ligand binding interactions may give such a result, for example, either by inhibiting the receptor-ligand interaction by binding the target receptor or ligand (desirable reasons), or by sickening or killing cells, or wielding other undefined effects (undesirable reasons).
  • chemoattractant receptor antagonists fail to identify all clinically important molecules, a consequence of false negative signals. False negatives mean that clinically important molecules are undetected and remain undiscovered. For example, a molecule that permits chemoattractant receptor ligand- - chemoattractant receptor binding, but inhibits chemoattractant receptor signaling, will be hidden in an initial screen for inhibitors of ligand binding.
  • chemokines a group of more than 40 small peptides (generally 7-10 kDa in size)
  • Chemokine-receptor binding is linked to G-protein-coupled signaling cascades to mediate chemoattractant and chemostimulant signaling functions.
  • Inappropriate chemokine signaling can either promote infections when not properly triggered (Forster et al., 1999) or lead to diseases associated with defective chemokine signaling, including asthma, allergic diseases, multiple sclerosis, rheumatoid arthritis, and atherosclerosis (reviewed in Rossi and Zlotnick, 2000). Because chemokines play pivotal roles in inflammation and lymphocyte development, the ability to specifically manipulate their activity will have enormous impact on ameliorating and halting diseases that currently have no satisfactory treatment. Chemokine receptor antagonists can be used to obviate the generalized and complicating effects of costly immunosuppressive pharmaceuticals in transplant rejection (reviewed in DeVries et al., 1999).
  • chemoattractant receptor antagonists such as those for chemokine receptors
  • an assay that weeds out false signals by testing both chemoattractant receptor binding and a biological function would hasten drug development.
  • the invention provides methods for identifying a chemoattractant receptor antagonist.
  • a cell having a chemoattractant receptor is incubated with a candidate antagonist in the presence of an excess of optimal ligand concentration for the chemoattractant receptor, and then cell migration is assayed. Cell migration indicates that the candidate antagonist is an antagonist.
  • the invention provides methods for identifying a chemokine receptor antagonist.
  • a cell expressing a chemokine receptor is incubated with a candidate antagonist in the presence of an inhibitory concentration of chemokine ligand, and then cell migration is assayed. Cell migration indicates that the candidate antagonist is an antagonist.
  • the invention provides methods for identifying a chemokine receptor antagonist.
  • a candidate antagonist of a chemokine receptor is first identified in a conventional assay.
  • the candidate antagonist is incubated with a chemokine receptor bearing cell in the presence of inhibitory concentration of ligand, and then cell migration is assayed. Cell migration confirms that the candidate antagonist is an antagonist.
  • RAM reversed-activation of migration
  • the methods of the invention include:
  • Cell migration is used to identify the candidate antagonist as an antagonist.
  • the method may further comprise a "pre-step” in which the concentration of a chemoattractant ligand (such as a chemokine) that inhibits cell migration is determined, the "inhibitory concentration" of a ligand for a chemoattractant receptor. Additional steps may be added, depending on the type of cell or agent being used, the assay, etc.
  • a chemoattractant ligand such as a chemokine
  • RAM assay Another advantage of the RAM assay is that the identified antagonists are more likely to be therapeutically useful than those identified in conventional assays.
  • a therapeutic chemoattractant receptor antagonist is specific for that receptor, exerting its effect through the receptor. Such an antagonist reduces the effective affinity between the chemoattractant and the receptor without compromising the physical integrity of the cell or completely disrupting the downstream signaling events leading to migration.
  • a false positive identified in a conventional assay lacks at least one of these characteristics.
  • a receptor antagonist that reduces the effective affinity of a chemoattractant for a receptor allows the ligand to behave like a ligand with lower affinity.
  • the bell-shape curve first observed in the absence of antagonists, shifts to the right in the presence of increasing concentrations of antagonist (see e.g., Figure 3). This is one possible explanation for the success of the present invention. The inventors do not intend to be limited by this proposal.
  • a "cell migration assay” tests the capacity of a cell to migrate in response to a signal.
  • an “inhibitory concentration" of a chemoattractant is one that inhibits cell migration. This concentration is greater than one that activates cell migration.
  • a "chemoattractant receptor” is a receptor that binds a chemoattractant ligand, inducing cell migration.
  • a chemokine receptor is a chemoattractant receptor whose chemoattractant ligand is at least one chemokine.
  • the RAM assay is illustrated using chemokines and chemokine receptors.
  • any chemoattractant and chemoattractant receptor that induces cell migration may be used.
  • Table A shows some examples of known chemoattractant receptors and some of their ligands.
  • chemoattractant receptors and exemplary ligands 1 Receptors examples of Iigands 2 BLT1 Leukotriene B4 PDGFR Platelet-Derived Growth Factor FPR fMLP FPRL1 Unknown FMLP receptor-like receptor Unknown CRTH2 prostaglandin D2 C3aR C3a C5aR C5a Noci-R Nociceptin EDG family Sphingosine 1-phosphate CB1 Cannabinoids VEGFR Vascular endothelial growth factor EGFR Epidermal growth factor FGFR Fibroblast growth factor P2Y receptor P2Y CTR Calcitonin CRLR Calcitonin gene-related peptide (CGRP) Receptor Examples of ligands 2 Histamine receptor Histamine Thrombin receptor Thrombin TrkB Brain-derived neurotrophic factor (BDNF) 1 This list of chemoattractant receptors is not meant to be exhaustive. 2 Only examples of some ligands for Iigand
  • a chemokine-bearing cell is incubated with a candidate antagonist and then contacted with an inhibitory concentration of a ligand for the target chemokine receptor. The ability of the cell to migrate is then assayed. If the cell migrates in the presence of a candidate antagonist in the RAM assay, then a positive signal has been observed.
  • Antagonist includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity, such as cell migration.
  • agonist includes any molecule that mimics a biological activity of molecule, such as a chemokine.
  • Molecules that can act as agonists or antagonists include small organic molecules, macromolecules, antibodies or antibody fragments, fragments or variants of chemokines, peptides, etc.
  • a “candidate antagonist” is a compound that is being tested for antagonist activity; likewise, a “candidate agonist” is a compound that is being tested for agonist activity.
  • Any cell migration assay format may be used, such as the ChemoTx® system (NeuroProbe, Rockville, MD) or any other suitable device or system (Bacon et al., 1988; Penfold et al., 1999).
  • these cell migration assays work as follows. After harvesting and preparing the cells bearing the active target chemokine receptor, the cells are mixed with candidate antagonists. The mixture is placed into the upper chamber of the cell migration apparatus. To the lower chamber, an inhibitory concentration of chemokine ligand is added. The migration assay is then executed, terminated, and cell migration assessed.
  • the solution of the inhibitory concentration of chemokine ligand is added to the lower chamber (6, Figure 4) of a cell migration apparatus, and the cell suspension is placed into the upper chamber (4, Figure 4) that is separated by a porous membrane (5, Figure 4).
  • the cells are incubated under culture conditions (37°C for human cells) for 60 to 180 minutes in a humidified tissue culture incubator. The incubation period depends on the cell type and if necessary, can be determined empirically.
  • the assay is terminated.
  • non-migrating cells on the upper chamber of the apparatus are removed, using a rubber scraper or other manual method; enzymatically or chemically, e.g., EDTA and EGTA solutions.
  • the membrane (5, Figure 4) that separates the two chambers is then removed from the apparatus and rinsed with Dulbecco's phosphate buffered saline (DPBS) or water. The number of cells that migrate into the lower chamber is then determined.
  • DPBS Dulbecco's phosphate buffered saline
  • the concentration of candidate antagonist to be screened in RAM assays may range from sub-nanomolar to millimolar. Screening a collection of small molecule compounds (such as a library synthesized by combinatorial chemistry), the concentration of candidate antagonists is typically about 1-20 ⁇ M.
  • “Compound” includes small inorganic and organic molecules, macromolecules, peptides, proteins, polypeptides, nucleic acids, and antibodies.
  • a dose response of cell migration to a chemokine ligand can be performed to define the inhibitory concentrations of a chemokine ligand.
  • Any standard method for determining dose response curves can be used. One such method includes harvesting cells expressing the target chemokine receptor, adding the cells to a cell migration device in the presence of increasing amounts of chemokine, measuring cell migration, plotting cell migration versus chemokine concentration, and then calculating from the graph those chemokine concentrations that inhibit cell migration.
  • a conventional cell migration assay such as the ChemoTx® system (NeuroProbe, Rockville, MD) or any other suitable device or system (Bacon et al., 1988; Penfold et al., 1999) may be used.
  • ChemoTx® system NeuroProbe, Rockville, MD
  • any other suitable device or system such as the ChemoTx® system (NeuroProbe, Rockville, MD) or any other suitable device or system (Bacon et al., 1988; Penfold et al., 1999) may be used.
  • To obtain a dose response curve cells expressing the target receptor are gathered.
  • a chemokine ligand is prepared in a concentration series by serial dilution in a buffer. The concentration range is typically between 0.1 nM and 10 mM, but will vary with ligand.
  • solutions of the various chemokine ligand concentrations are added to the lower chamber (6, Figure 4) of a cell migration apparatus, and the cell suspension is placed into the upper chamber ( 4 , Figure 4) that is separated by a porous membrane ( 5 , Figure 4).
  • the cells are incubated under culture conditions (37°C for human cells) for 60 to 180 minutes in a humidified tissue culture incubator. The incubation period depends on the cell type and if necessary, can be determined empirically.
  • non-migrating cells on the upper chamber of the apparatus are removed, using a rubber scraper or other manual method; enzymatically or - chemically, e.g. , EDTA and EGTA solutions.
  • the membrane ( 5 , Figure 4) that separates the two chambers is then removed from the apparatus and rinsed with Dulbecco's phosphate buffered saline (DPBS) or water. The number of cells that migrate into the lower chamber is then determined.
  • DPBS Dulbecco's phosphate buffered saline
  • Y-axis is then plotted against the log(chemokine concentration) (X-axis); a bell-shaped curve is observed (Figure 2; see Examples). From this plot ( Figure 2), the lowest concentration of chemokine that inhibits cell migration can be determined.
  • a second Y-axis (y 2 , 1 , Figure 2) can be drawn through the bell curve, intersecting at its apex (maximal cell migration) and the corresponding value on the X-axis.
  • Those concentrations to the left of the Y 2 -axis (lower) are stimulatory ( 2 , Figure 2); those to the right (higher) are inhibitory ( 3 , shaded region, Figure 2).
  • concentrations are the “inhibitory concentrations” for cell migration (chemotaxis). For example, to determine the concentration at with migration is inhibited by 90% of the maximum (to the right of the Y 2 -axis, the “inhibitory” concentrations), the value corresponding to 10% of maximal cell migration on the Y-axis is located. If the maximal cell migration signal is, e.g. , 3.5 x 10 4 cells, 10% thereof would be 350 (3.5 x 10 4 x 0.1). The inhibitory ligand concentration is then determined by locating the corresponding X-axis coordinate. Preferably, the level of inhibition is 50%, 60%, 70% or 80% of maximal cell migration.
  • the level of inhibition is 90% or even more preferably 95% or 100% inhibition as compared to the maximal signal for migration.
  • the determined chemokine concentration varies and depends on the nature of the receptor, the chemokine ligand and the target cell. Varying the degree of chemotactic inhibition can be used to modulate the sensitivity of the RAM assay.
  • RAM assays can be performed in conjunction with any other assay used to screen for chemokine receptor antagonists. Not only is the RAM format useful as a primary HTS step, but it also provides a confirmatory or secondary assay for candidate antagonists identified in other assays. For example, a HTS method that measures Ca 2+ mobilization, including those based on the FLIPR TM system (Molecular Devices Corp., Sunnyvale, CA) or other reporter-based methods which assay increases in free intracellular Ca 2+ levels, can be used as a primary assay. RAM assays can be used to confirm such candidates, or vice-versa. As a secondary assay, RAM would discriminate those candidate antagonists that exert non-specific effects. When RAM assays are used with other HTS methods, a means for discriminating true hits from non-specific blockers is provided.
  • the RAM assay can be applied to any other assay format measuring cell migration or receptor activation, including methods that do not require migration of cells across a porous membrane. More useful technologies offering higher throughput and lower cost may be developed based on use of the RAM concept.
  • Cells expressing a target chemokine receptor (or chemoattractant receptor) for use in the RAM assay may be gathered by a variety of methods, for example by centrifugation after collection from a subject or release from culture, and then resuspended in a buffer at an appropriate density, depending on cell type and cell size.
  • Convenient cell concentrations range from about 1 x 10 6 to 1 x 10 7 cells/ml; often about 2.5 x 10 6 cells/ml is suitable.
  • Cells that can be assayed in the RAM format include all those that express at least one chemokine receptor on the cell surface, such as human monocytes, or other cells engineered to express recombinant chemokine receptors and are competent to activate cell migration.
  • chemokine receptors and some of their ligands are shown in Table B. Examples of chemokine receptors include, but are not limited to, the CXC class, e.g.
  • CXCR1, CXCR2, CXCR3, CXCR4, CXCR5 the CC class, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11; the CX3CR class, such as CX3CR1 and the XCR class, such as XCR1.
  • non-chemokine chemoattractant receptor is formyl peptide receptor like protein 1 (FPRL1); the ligands for which are w-peptide1 and w-peptide2 (Klein et al., 1998). Also see Table A for other examples.
  • FPRL1 formyl peptide receptor like protein 1
  • Chemokines that can be used in the RAM assay include all known chemokines.
  • chemokines include, but are not limited to, IL-8, GCP-2, Gro ⁇ , Gro ⁇ , Gro ⁇ , ENA-78, PBP, MIG, IP-10, I-TAC, SDF-1 (PBSF), BLC (BCA-1), MIP-1 ⁇ , MIP-1 ⁇ , RANTES, HCC-1, -2, -3, and -4, MCP-1, -2, -3, and -4, eotaxin-1, eotaxin-2, TARC, MDC, MIP-3 ⁇ (LARC), MIP-3 ⁇ (ELC), 6Ckine (LC), I-309, TECK, lymphotactin, fractalkine (neurotactin), TCA-4, Exodus-2, Exodus-3 and CK ⁇ -11.
  • Chemokine receptor/ligand combinations include those associated with inflammatory disorders, infectious diseases and transplant rejection. Such combinations include CX3CR1/fractalkine (transplantation), CCR5/MIP-1 ⁇ , MIP-1 ⁇ , or RANTES (HIV), CXCR4/SDF-1 (HIV); and CCR7/MIP-3 ⁇ , ELC or 6Ckine LC (inflammatory or allergic diseases, e.g. asthma, multiple sclerosis, etc.).
  • Any molecule or compound can be screened for chemokine receptor antagonist activity.
  • Compounds that inhibit chemokine receptor/ligand activities, such as activating cell migration or modulating intracellular Ca 2+ concentrations are candidate antagonists.
  • Such molecules that may exert such antagonistic effects include small molecules that bind to chemokine receptors or their ligands.
  • small molecule antagonists include small peptides, peptide-like molecules, preferably soluble and synthetic non-peptidyl organic or inorganic compounds.
  • Other potential antagonist molecules include nucleic acids such as aptamers and antibodies. These molecules may be collected into various libraries can be quickly screened for novel chemokine receptor antagonists using the RAM assay.
  • antibody that inhibits chemotactic cell migration
  • antibody antagonists include polyclonal, monoclonal, single-chain, anti-idiotypic, chimeric Abs, or humanized versions of such Abs or fragments. Abs may be from any species in which an immune response can be raised. Humanized Abs are exceptionally well-adapted for treatment of diseases and represent attractive candidate antagonists (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988); (U.S. Patent No. 4816567, 1989). Such antibodies may bind to chemokine receptors to inhibit cell migration.
  • a potential antagonist or agonist may be a closely related protein, for example, a mutated form of a chemokine receptor ligand or other protein that recognizes a chemokine receptor interacting protein, but imparts no effect, thereby competitively inhibiting chemokine receptor action.
  • Aptamers are short oligonucleotide sequences that can be used to recognize and specifically bind almost any molecule, such molecules may also act antagonistically.
  • the systematic evolution of ligands by exponential enrichment (SELEX) process (Ausubel et al., 1987; Ellington and Szostak, 1990; Tuerk and Gold, 1990) is powerful and can be used to find such aptamers.
  • Aptamers have many diagnostic and clinical uses, including as antagonists. In addition, they are inexpensive to manufacture and can be easily applied in a variety of formats, including administration in pharmaceutical compositions, bioassays, and diagnostic tests (Jayasena, 1999).
  • the RAM assay can also be used as a screen to isolate aptamers de novo.
  • Quantifying migratory cells may be accomplished by a large variety of available methods, such as those that assay the amount of DNA, (e.g., the CyQuant Cell Proliferation Kit (Molecular Probes)) and then assaying the generated signal, such as fluorescence. Other methods include counting the cells using a microscope, or labeling cells with a suitable detectable marker, such as dyes (such as Calcein AM (NeuroProbe) or the many labels available from Molecular Probes (Eugene, OR)) or radioactive labeling (e.g. cell surface iodination with 135 I, protein synthesis labeling with 35 S-methionine/ 35 S-cysteine or nucleic acid labeling with 3 H).
  • a detectable marker such as dyes (such as Calcein AM (NeuroProbe) or the many labels available from Molecular Probes (Eugene, OR)
  • radioactive labeling e.g. cell surface iodination with 135 I, protein synthesis labeling with 35 S
  • Buffers that may be used to prepare the various solutions include cell culture media, although serum or other growth and chemotactic factors may be removed so that the results in a cell migration assay are not confounded and can be mostly attributable to the chemokine-chemokine receptor interaction.
  • a protein may be added to support the cells, such as various albumins, including bovine serum albumin.
  • Optimal media selection depends on the cell type; that media used to culture the cells usually represents a preferred option.
  • Suitable culture media include Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium Eagle (MEM), Basal Medium Eagle (BME), Click's Medium, L-15 Medium Leibovitz, McCoy's 5A Medium, Glasgow Minimum Essential Medium (GMEM), NCTC 109 Medium, Williams' Medium E, RPMI-1640, and Medium 199.
  • IMDM Iscove's Modified Dulbecco's Medium
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium Eagle
  • BME Basal Medium Eagle
  • Click's Medium L-15 Medium Leibovitz
  • McCoy's 5A Medium Glasgow Minimum Essential Medium (GMEM)
  • NCTC 109 Glasgow Minimum Essential Medium
  • Williams' Medium E Williams' Medium E, RPMI-1640, and Medium 199.
  • a medium specifically developed for a particular cell type/line or cell function e.g.
  • Madin-Darby Bovine Kidney Growth Medium Madin-Darby Bovine Kidney Maintenance Medium, various hybridoma media, Endothelial Basal Medium, Fibroblast Basal Medium, Keratinocyte Basal Medium, and Melanocyte Basal Medium are also useful.
  • a protein-reduced or free and/or serum free medium and/or chemically defined, animal component free medium may be used, e.g., CHO, Gene Therapy Medium or QBSF Serum-free Medium (Sigma Chemical Co.; St.
  • DMEM Nutrient Mixture F-12 Ham MCDB (105, 110, 131, 151, 153, 201 and 302), NCTC 135, Ultra DOMA PF or HL-1 (both from Biowhittaker; Walkersville, MD), may be used.
  • the media may be further supplemented with reagents that limit acidosis of the cultures, such as buffer addition to the medium (such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris), N-(2-hydroxyethyl)piperazine-N'3-propanesulfonic acid (EPPS or HEPPS), glyclclycine, N-2-hydroxyehtylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propane sulfonic acid (MOPS), piperazine-N,N'-bis(2-ethane-sulfonic acid) (PIPES), sodium bicarbonate, 3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid) TAPSO, (N), buffer
  • kits, containers, packs, or dispensers may be assembled into kits, containers, packs, or dispensers together with instructions for administration.
  • the different components of the composition may be packaged in separate containers and admixed immediately before use.
  • packaging of the components separately may permit long-term storage without losing the active components' functions.
  • a kit may include a cell migration apparatus, a chemokine receptor-bearing cell and a solution comprising an inhibitory migratory concentration of chemokine for the chemokine receptor bearing cell.
  • the solution may be supplied lyophilized.
  • the reagents included in the kits can be supplied in containers of any sort such that the life of the different components are preserved, and are not adsorbed or altered by the materials of the container.
  • sealed glass ampules may contain lyophilized chemokine or a buffer that has been packaged under a neutral, non-reacting gas, such as nitrogen.
  • Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold reagents.
  • suitable containers include simple bottles that may be fabricated from similar substances as ampules, and envelopes, that may consist of foil-lined interiors, such as aluminum or an alloy.
  • Containers include test tubes, vials, flasks, bottles, syringes, or the like.
  • Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
  • Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix; for example, lyophilized chemokine in one compartment, and a buffer or water in the other.
  • Removable membranes may be glass, plastic, rubber, etc.
  • Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
  • chemoattractant receptor and ligands used to illustrate the invention are chemokine receptors and chemokines.
  • any chemoattractant ligand for any chemoattractant receptor may be used. For examples, see Table A.
  • Examples 1, 2 and 4 demonstrate the effectiveness of the RAM assay, testing specific and non-specific antagonists of CXCR4 as discovered in conventional assays.
  • Example 3 demonstrates the broad applicability of chemoattractant receptors by examining three chemokine receptors.
  • a conventional cell migration assay was used (Bacon et al., 1988; Penfold et al., 1999).
  • Cells were harvested by centrifugation and then resuspended in cell migration buffer (Hank's balanced salt solution (HBSS)/0.1% bovine serum albumin (BSA) at 2.5 x 10 6 cells/ml.
  • HBSS Horbal's balanced salt solution
  • BSA bovine serum albumin
  • SDF-1 was prepared in a concentration series (0.1 nM to 10 mM) by serial dilution in cell migration buffer. At low concentrations, SDF activates cell migration of CXCR4-bearing activated lymphocytes.
  • SDF ligand was loaded in the bottom chamber of a ChemoTx ® cell migration apparatus (5 ⁇ m pore polycarbonate polyvinylpyrrolidone-coated filters (Neuroprobe; Gaithersburg, MD); 29 ⁇ l/well) and 20 ⁇ l of cell suspension was placed in the upper chamber. The cells were incubated at 37°C for 150 minutes. The assay was terminated by removing the cells from the upper chamber and membrane surface using a rubber scraper. The cells that migrated to the lower chamber were quantified by the CyQuant assay (Molecular Probes; Eugene, OR), a fluorescent dye method that measures nucleic acid content.
  • CyQuant assay Molecular Probes; Eugene, OR
  • chemokine concentration (X-axis) is plotted against relative fluorescent units (RFUs), correlating to the number of cells migrating (Y-axis) ( Figure 2).
  • REUs relative fluorescent units
  • chemokine receptors are identified by their ability to activate migration of cells that are incubated with inhibitory chemokine concentrations.
  • the viral chemokine, vMIP-II was used as a CXCR4 antagonist.
  • vMIP-II binds with high affinity to CXCR4, blocking receptor signaling and inhibiting cell migration, competing with CXCR4's usual ligand, SDF (Kledal et al., 1997). If CXCR4 expressing cells that are immobilized by inhibitory concentrations of SDF are activated to migrate in the presence of vMIP-II with increased migration, this result would verify the RAM assay principle.
  • a conventional cell migration assay was performed. In the conventional assay format, cell migration is inhibited by vMIP-II.
  • Activated lymphocytes expressing cell surface CXCR4 were harvested as in Example 1.
  • a concentration series of vMIP-II was first mixed with activated lymphocytes, and the solution then placed in the upper chamber of a ChemoTx ® cell migration apparatus (5 ⁇ m pore polycarbonate polyvinylpyrrolidone-coated filters (Neuroprobe), 20 ⁇ l/well); 29 ⁇ l of a 1 nM solution of SDF was placed in the lower chamber.
  • the cells were prepared as for the conventional assay, except the SDF concentration in the lower chamber was 1 ⁇ M. The cells were incubated at 37°C for 150 minutes. The assay was terminated by removing the cells from the upper chamber and membrane surface using a rubber scraper. The cells that migrated to the lower chamber were quantified by the CyQuant assay (Molecular Probes).
  • RAM assays were performed as described in Example 2, except previously identified small molecule antagonists instead of vMIP-II were used, as well as additional cell types as described in Table 2.
  • Table 2 Experimental variables Antagonist 1 Receptor Ligand(s) Cells RAMAG-1 CXCR3 I-TAC (250 nM), IP-10 activated human lymphocytes RAMAG-2 CXCR4 SDF-1 CXCR4-expressing MOLT-4 cells (human T lymphoblast; American Type Tissue Collection (ATCC); Manassas, VA) RAMAG-3 CCR1 MIP-1 ⁇ THP-1 cells (human monocytic; ATCC) 1 As defined using a conventional cell migration assay and further independently confirmed. 2 Inhibitory concentrations determined as in Example 1.
  • RAMAG-2 activated cell migration at 5 ⁇ M ( Figure 5, B). As was observed with RAMAG-1, further activation of migration was seen as the RAMAG-2 concentration increased to 10 ⁇ M.
  • the RAM assay distinguishes between non-specific and specific antagonists of chemoattractant receptors, such as chemokine receptors.

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Claims (20)

  1. Verfahren zum Identifizieren eines Antagonisten eines Rezeptors für chemische Lockstoffe, umfassend:
    Inkubieren einer Zelle, die einen Rezeptor für chemische Lockstoffe umfasst, mit einem Kandidaten für einen Antagonisten;
    Inkontaktbringen der Zelle mit einer hemmenden Konzentration eines Liganden für den Rezeptor für chemische Lockstoffe; und
    Untersuchen der Zellwanderung, wobei die Zellwanderung den Kandidaten für einen Antagonisten als einen Antagonisten identifiziert.
  2. Verfahren nach Anspruch 1, wobei der Kandidat für einen Antagonisten ein kleines Peptid, ein peptidähnliches Molekül, eine organische Verbindung, bei der es sich nicht um ein Peptid handelt, eine anorganische Verbindung, eine Nukleinsäure oder ein Antikörper ist.
  3. Verfahren nach Anspruch 1, wobei die hemmende Konzentration eines Liganden für den Rezeptor für chemische Lockstoffe die Zellwanderung um mehr als oder gleich 50 % der maximalen Liganden-aktivierten Zellwanderung hemmt.
  4. Verfahren nach Anspruch 1, wobei die hemmende Konzentration eines Liganden für den Rezeptor für chemische Lockstoffe die Zellwanderung um mehr als oder gleich 95 % der maximalen Liganden-aktivierten Zellwanderung hemmt.
  5. Verfahren nach Anspruch 1, wobei die hemmende Konzentration eines Liganden für den Rezeptor für chemische Lockstoffe die Zellwanderung um 100 % der maximalen Liganden-aktivierten Zellwanderung hemmt.
  6. Verfahren nach Anspruch 1, wobei der Rezeptor für chemische Lockstoffe den Rezeptor FPRL1 umfasst und der chemische Lockstoff einen Liganden für FPRL1 umfasst.
  7. Verfahren nach Anspruch 6, wobei der Ligand für FPRL1 aus der Gruppe, die aus W-Peptid1 und W-Peptid2 besteht, ausgewählt ist.
  8. Verfahren zum Identifizieren eines Antagonisten eines Chemokinrezeptors, umfassend:
    Inkubieren einer Zelle, die einen Chemokinrezeptor umfasst, mit einem Kandidaten für einen Antagonisten;
    Inkontaktbringen der Zelle mit einer hemmenden Konzentration eines Liganden für den Chemokinrezeptor; und
    Untersuchen der Zellwanderung, wobei die Zellwanderung den Kandidaten für einen Antagonisten als einen Antagonisten identifiziert.
  9. Verfahren nach Anspruch 8, wobei der Kandidat für einen Antagonisten ein kleines Peptid, ein peptidähnliches Molekül, eine organische Verbindung, bei der es sich nicht um ein Peptid handelt, eine anorganische Verbindung, eine Nukleinsäure oder ein Antikörper ist.
  10. Verfahren nach Anspruch 8, wobei es sich bei dem Chemokinrezeptor um CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1 oder XCR1 handelt.
  11. Verfahren nach Anspruch 8, wobei das Chemokin aus der Gruppe, die aus Liganden für CCR, CXCR und CX3CR besteht, ausgewählt ist.
  12. Verfahren nach Anspruch 11, wobei es sich bei dem Chemokin um IL-8, GCP-2, Gro α, Gro β, Gro γ, ENA-78, PBP, MIG, IP-10, 1-TAC, SDF-1, BLC, MIP-1α, MIP-1β, RANTES, HCC-1, HCC-2, HCC-3, HCC-4, MCP-1, MCP-2, MCP-3, MCP-4, Eotaxin-1, Eotaxin-2, TARC, MDC, MIP-3α, MIP-3β, 6Ckine, I-309, TECK, Lymphotactin, Fractalkin, TCA-4, Exodus-2, Exodus-3 oder CKβ-11 handelt.
  13. Verfahren nach Anspruch 8, wobei der Chemokinrezeptor CCR5 umfasst und das Chemokin MIP-1α, MIP-1β oder RANTES umfasst.
  14. Verfahren nach Anspruch 8, wobei der Chemokinrezeptor CXCR4 umfasst und das Chemokin SDF-1 umfasst.
  15. Verfahren nach Anspruch 8, wobei der Chemokinrezeptor CCR9 umfasst und das Chemokin TECK umfasst.
  16. Verfahren nach Anspruch 8, wobei der Chemokinrezeptor CCR10 umfasst und das Chemokin CTACK umfasst.
  17. Verfahren nach Anspruch 8, wobei die hemmende Konzentration eines Liganden für den Chemokinrezeptor die Zellwanderung um mehr als oder gleich 50 % der maximalen Liganden-aktivierten Zellwanderung hemmt.
  18. Verfahren nach Anspruch 8, wobei die hemmende Konzentration eines Liganden für den Chemokinrezeptor die Zellwanderung um mehr als oder gleich 95 % der maximalen Liganden-aktivierten Zellwanderung hemmt.
  19. Verfahren nach Anspruch 8, wobei die hemmende Konzentration eines Liganden für den Chemokinrezeptor die Zellwanderung um 100 % der maximalen Liganden-aktivierten Zellwanderung hemmt.
  20. Verfahren zum Identifizieren eines Antagonisten eines Chemokinrezeptors, umfassend:
    Identifizieren eines Kandidaten für einen Antagonisten eines Chemokinrezeptors in einem herkömmlichen Assay;
    weiter umfassend einen zweiten Schritt, umfassend:
    Inkubieren einer Zelle, die den Chemokinrezeptor umfasst, mit dem Kandidaten für einen Antagonisten;
    Inkontaktbringen der Zelle mit einer hemmenden Konzentration eines Liganden für den Chemokinrezeptor; und
    Untersuchen der Zellwanderung, wobei die Zellwanderung den Kandidaten für einen Antagonisten als einen Antagonisten identifiziert.
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